Microscopy denotes a class of analytical techniques involving the imaging of samples such as, for example, biological material (e.g., cells, tissue, etc.) and non-biological material (e.g., particulate matter). Known light-based (optical) microscopic techniques include, for example, bright-field microscopy, dark-field microscopy, confocal microscopy, and selective plane illumination microscopy (SPIM). A light-based microscopic technique may be enhanced by inducing fluorescence in the sample being imaged. Moreover, some microscopes are capable of three-dimensional (3D) imaging of a sample, typically by scanning the axial depth of focus through the sample to acquire multiple images (a focal stack) and subsequently processing the images to generate a 3D image. This post-acquisition processing typically entails heavy computation, including the use of 3D deconvolution algorithms, registration algorithms, etc.
3D fluorescence imaging of biological samples is typically performed by confocal microscopy or SPIM. Confocal microscopy can deliver brilliant and highly resolved images, but is highly time consuming and requires complex and thus costly system hardware. SPIM is potentially faster and cheaper, but needs special sample preparation and holders. SPIM is described by Huisken et al., Selective plane illumination microscopy techniques in developmental biology, Development, Vol. 136(12), p. 1963-1975 (2009), the content of which is incorporated by reference herein in its entirety. Recently, light-field imaging is being proposed as a 3D microscopic imaging method. Light-field imaging captures information regarding both the intensity of light in a scene and the direction of travel of the light rays, as compared to capturing just light intensity in the case of traditional microscopes. Thus far, however, experimental work has shown that light-field imaging produces an unacceptable amount of image artifacts and background noise. Light-field technology is described in U.S. Pat. No. 7,936,392; U.S. Pat. No. 8,717,489; Levoy et al., Light Field Microscopy, ACM Transactions on Graphics, Vol 25(3), p. 1-11, Proc. SIGGRAPH (2006); and Cohen et al., Enhancing the performance of the light field microscope using wavefront coding, OPTICS EXPRESS, Vol. 22, No. 20 (2014); and Broxton et al., Wave optics theory and 3-D deconvolution for the light field microscope, OPTICS EXPRESS, Vol. 21, No. 21 (2013); the contents of each of which are incorporated by reference herein in their entireties.
There is an ongoing need for microscopes and methods for 3D microscopic imaging. Particularly for studies and analyses of complex and functional biological samples such as organoids and spheroids, a fast microscopic imaging method resolving 3D information is needed.